Inhibition of ALDH2 by quercetin glucuronide suggests a new hypothesis to explain red wine headaches

The consumption of red wine induces headaches in some subjects who can drink other alcoholic beverages without suffering. The cause for this effect has been attributed to a number of components, often the high level of phenolics in red wine, but a mechanism has been elusive. Some alcohol consumers exhibit flushing and experience headaches, and this is attributed to a dysfunctional ALDH2 variant, the enzyme that metabolizes acetaldehyde, allowing it to accumulate. Red wine contains much higher levels of quercetin and its glycosides than white wine or other alcoholic beverages. We show that quercetin-3-glucuronide, a typical circulating quercetin metabolite, inhibits ALDH2 with an IC50 of 9.6 µM. Consumption of red wine has been reported to result in comparable levels in circulation. Thus, we propose that quercetin-3-glucoronide, derived from the various forms of quercetin in red wines inhibits ALDH2, resulting in elevated acetaldehyde levels, and the subsequent appearance of headaches in susceptible subjects. Human-subject testing is needed to test this hypothesis.

the normal levels) after alcohol consumption [9][10][11][12] .This high level of acetaldehyde causes facial flushing, headache, tachycardia, and nausea, similar to disulfiram treatment 9 .This similarity between these two scenarios and the accumulation of acetaldehyde suggests a relationship between acetaldehyde accumulation and headache caused by alcoholic beverages.
In a meta-analytic review on alcohol use disorders (AUDs) in primary headache, 28% of studies endorsed red wine as the trigger, followed by spirits (14%), white wine (10%) and sparkling wine/beer (10%) 13 .Red wine headache (RWH) does not require excessive amounts of wine as a trigger.In most cases, the headache is induced in 30 min to 3 h after drinking only one or two glasses of wine 3 .Wine constituents such as biogenic amines, sulphites, phenolic flavonoids, or tannins have been reported as the possible cause of wine headaches 3,4,[14][15][16][17][18] .Nevertheless, no chemical constituent has been clearly implicated as the primary trigger of red wine headache (RWH) nor has a mechanism for eliciting the headache been proposed.
The higher amount of phenolic compounds, especially flavonoids, in red wine, tenfold compared to white wine, make them a primary contender responsible for RWH 16 .However, phenolics and high phenolic foods have not been linked to headache.Interestingly, some red wine phenolics such as quercetin, and resveratrol have been reported to affect the activity of ALDH 19,20 .Quercetin was reported as potent inhibitor of cytosolic aldehyde dehydrogenase (ALDH1) at low concentration (< 1 mM) of acetaldehyde and cofactor NAD+ 19 .However, these studies were conducted to relate the activity of the ALDH with anticancer properties and birth defects 19,21 and not with acetaldehyde metabolism.Further, none of the mentioned studies reported the effect of quercetin on mitochondrial aldehyde dehydrogenase (ALDH2).A study by Keung and Vallee 22 reported the effect of several flavonoids including quercetin on both ALDH1 and ALDH2 activity.They found no effect of quercetin and other red wine flavonoids (kaempferol, rutin and myricetin) on both aldehyde dehydrogenases.But the enzyme assay in the study was performed at pH 9.5, and thus are of marginal significance at physiological pH.In another study Orozco et al. 23 reported inhibition of yeast cytosolic and mitochondrial ALDH by quercetin during red wine fermentation.Thus, evaluation of red wine flavonoids on ALDH2 activity, with possible effect on acetaldehyde metabolism might provide clues to RWH.
In order to assess this possible mechanism for RWH, we evaluated the inhibition of mitochondrial ALDH2 by red wine flavonoids, especially quercetin derivatives, using an in vitro enzymatic assay.

In vitro inhibitory effect of red wine phenolics/flavonoids on ALDH2
The inhibitory effect of selected wine phenolics/flavonoids (at 20 µM concentration) was evaluated in vitro on the inhibition of ALDH2 (in %, Table 1).Among the selected compounds, quercetin glucuronide (compound 9), a liver metabolite of quercetin, showed the highest inhibitory activity (78.69 ± 1.21%) whereas the least inhibition was observed for epicatechin (0.34 ± 0.12%).For all other compounds (1-8, 10-12), the inhibition activity ranged from 14.77 ± 0.39 to 27.69 ± 0.61%, suggesting a low to moderate inhibition effect of wine flavonoids on ALDH2.
Further, it was observed that glycosylated forms of quercetin (compounds 3-7) exhibited lower inhibitory activity than the aglycone (compounds 1-2).However, the methoxylated quercetin, tamarixetin did not differentially inhibit the enzyme compared to quercetin.
Quercetin-3-glucuronide was selected as a potent inhibitor for further study based on the inhibition screening assay.The ALDH2 inhibition by different concentrations of quercetin-3-glucuronide (5-20 µM) is depicted in Fig. 1.Quercetin-3-glucuronide has an IC 50 of 9.62 µM, a stronger inhibition of ALDH2 than the aglycone, quercetin (IC 50 of 26.50 µM).However, the inhibition by quercetin-3-glucuronide is lower than the drug disulfiram (IC 50 of 1.45 µM, data not shown), used clinically to inhibit the enzyme.Table 1.Inhibition of ALDH2 activity by selected red wine phenolics using the QuantiChrom™ kit.Values are mean ± SD for n = 3 for each phenolic compound.Different alphabet in superscript represent that the values are significantly different at p < 0.05.

Discussion and conclusion
Flavonols, such as quercetin, myricetin, and kaempferol, are present in wine, either as glycosides or the aglycone.
In two studies, the total flavonol content in white wines (mean value: traces to 7 mg/L) are almost ten-fold lower than the red wines (mean value: 4-93 mg/L) 24,25 .Burns et al. 26 reported total quercetin and free quercetin ranging from 5 to 104.7 µmoles and 1.8-41.9µmoles in 16 red wines from different wine countries, varieties and vinification methods.Many other studies have reported quercetin contents in Italian 27,28 , Australian 24 , Spanish 29 , and Bulgarian 30 wines.It is evident from the reports that there is a variation in the quercetin content in the red wines, and most investigations evaluated a relatively small number of samples (n < 20).Quercetin-3-glucuronide is also reported in red wines as one of the major quercetin glycosides 24,31,32 .Castillo-Muñoz et al. 32 found quercetin-3 glucuronide in the range of 10.26 to 13.81 mg/L in different Vitis vinifera single cultivar red wines.Ghiselli et al. 31 observed 19 mg/L of quercetin-3-glucuronide in Italian Sangiovese wine.Jeffery et al. 24 found quercetin-3-glucuronide as the predominant and most stable glycoside, ranging from below detection level to 39.67 mg/L among 121 Australian red wines.
Studies have repeatedly noted that the level of quercetin is 4 to 8 times higher in sun exposed grape clusters than the shaded clusters 33,34 .Ritchey and Waterhouse 35 evaluated the total quercetin content in high-volume versus ultra-premium commercial Cabernet Sauvignon wines.The average total flavonols was four times higher in ultra-premium wines (202 mg/L) than in high-volume wines (53 mg/L).The study explained that vineyard practices (trellised vines, crop thinning, leaf removal) in the areas that produce ultra-premium wines would result in more sun exposure, which in turn, allows higher production of quercetin.But, the variations in levels arise not just from differences in grape composition induced by sun exposure, but also from wine-making techniques, including skin contact during fermentation, stabilization/fining procedures, and aging methods 26 .Quercetin absorption pathways in the gastrointestinal tract of humans and mammals are well understood 36 .Most quercetin absorption occurs in the small intestine, and a minor amount is absorbed in the stomach 36 .Quercetin aglycone and glucosides are not found in blood plasma upon absorption.In plasma, it appears as conjugates (78-79%), including quercetin-3-glucuronide, quercetin-3-sulfate, and methylated forms such as tamarixetin (10-13%) and isorhamnetin (8.5 to 11%) 36,37 .Numerous studies have been reported on quercetin bioavailability from foods and food extracts (onions, blackcurrants, bilberries, apples, red grapes, tomatoes), beverages (tea, fruit juices, coffee, cocoa, and wine) and supplements in the form of a solution, powder, capsule, or tablet 36 .The quercetin derivatives or catabolites are generally measured in blood and urine to assess their bioavailability.de Vries et al. 38 compared the bioavailability of flavonol from 750 mL red wine (47.0 µmole quercetin), 50 g fried onion (52.6 µmole quercetin), and 375 mL black tea (45.3 µmole quercetin) in 12 healthy men.They observed that with comparable "doses" the plasma quercetin (including conjugates) level after red wine consumption (0.026 µM) is half of that of onions (0.053 µM).The study also found variation in plasma quercetin levels within persons of 10% and between persons of 10-20%.In another study, Spaak et al. 39 , the consumption of 2 drinks of Pinot noir containing 37.7 µmole of quercetin resulted in a blood plasma total quercetin concentration of 2.74 µM.Goldberg et al. 40 obtained a blood plasma total quercetin level of 4.19 µM after consumption of white wine that contained a dose of 25 mg/70 kg body weight quercetin aglycone.The total concentration of quercetin and conjugates detected in blood plasma was 0.84 µM, i.e., 26.9% of the quercetin dose.
Hence, assuming the reported concentration of quercetin in wine as 346 µM, a standard drink of wine (147 mL) would have quercetin content of 50.6 µM.Thus, the blood plasma total quercetin concentration would be expected to be about 6 µM.Further, considering nearly 80% of the total quercetin in plasma corresponds to conjugated quercetin (mostly quercetin-3-glucuronide), the level in plasma would be 5 µM.Hence, based on our study, one standard drink of wine with 5 µM quercetin-3-glucuronide would result in nearly 37% of ALDH2 inhibition.The enzyme inhibition would result in acetaldehyde accumulation from the alcohol metabolism from wine, likely causing RWH, which otherwise is not evident in other foods (for instance, onions) with higher bioavailable quercetin but no alcohol.However, the actual inhibition will be higher or lower, depending on the concentration of other components, such as cofactors, buffers, etc.These results-suggest that RWH could be the result of ALDH2 inhibition by red wine quercetin metabolites.
Testing this hypothesis will require validation.An obvious experiment would be to compare wines having differing phenolic levels (particularly quercetin and total flavonols) with observed headache occurrences after ingestion, although there would be matrix differences.Controlling alcohol levels would be imperative.Another, simpler, experiment would be to provide RWH subjects with a quercetin supplement or placebo and a standard drink of vodka, to see if headaches result.
To probe the concept at a more fundamental level, other ideas come to mind.What concentration of circulating quercetin-3-glucuronide inhibit ALDH2 in vivo, yielding elevated levels of acetaldehyde?Are these levels correlated with the perception of a headache among RWH subjects?At the same concentrations, do RWH-susceptible subjects experience greater inhibition, and higher levels of acetaldehyde?Or, alternatively, do RWH subjects have a genetic tendency, to variations in ALDH2 isoforms (ALDH2*1 and ALDH2*2), to be more sensitive to acetaldehyde, and could this be related to the documented propensity of these subjects towards migraine headaches 3 .Also, genomic studies in these subjects could illuminate genetic polymorphism underlying this troublesome condition.
As noted above, there is considerable variation in quercetin levels among wines in the market.Hence, more detailed analysis of large samples of wines for quercetin levels could be helpful to guide wine drinkers who suffer RWH.But current measurement techniques are complex, so, a simple and fast detection method for flavanols or quercetin, such as a secondary spectral method, could provide a quick headache-potential assessment.
In conclusion, the present study provides an indication that the headache caused by red wine is due to the presence of quercetin and its glycosides, which upon metabolizing to quercetin glucuronide, inhibits ALDH2 enzyme activity.With the concurrent consumption of alcohol, the in vitro inhibition of ALDH2 by quercetin glucuronide would lead to an accumulation of toxic acetaldehyde, resulting in headaches.Further studies on human subjects are needed to verify this hypothesis.
Human recombinant ALDH2 enzyme was purchased from Sigma-Aldrich, USA.The enzyme purity was > 90% and have the biological activity of > 0.14 units/mL as mentioned in the manufacturer's certificate of analysis.The QuantiChrom™ aldehyde dehydrogenase inhibitor screening kit (EIAL-100) was procured from BioAssay Systems, Thermo-Fischer Scientific, USA for the quantitative determination of ALDH2 activity inhibition by phenolic compounds.

Determination of ALDH2 inhibition by phenolic compounds
The ALDH2 inhibition by selected phenolic compounds was measured using the QuantiChrom™ aldehyde dehydrogenase inhibitor screening kit (EIAL-100, BioAssay systems, USA) as per the manufacturer's instruction.In principle, the kit is based on enzymatic conversion of acetaldehyde to acetic acid and NADH by ALDH.The formed NADH in turn reduces a formazan reagent into a colored product, the absorbance of which measured at 565 nm, is proportional to the enzyme activity in the reaction.Briefly, the assay is performed in 96 well-plates by adding 0.1 U/mL of ALDH2, 20 µM phenolic compound in DMF to reaction mix comprising of assay buffer, NAD/MTT, diaphorase and 1× substrate provided in the kit.To the control, ALDH2 and solvent DMF without phenolic compound was added to the reaction mix as mentioned earlier.Blank reaction was prepared by adding 0.1 U/mL of ALDH2, solvent DMF without phenolic compound, assay buffer, NAD/MTT and diaphorase.The final volume of all the reactions was 100 µL.The reactions were incubated for 30 min at room temperature and the optical density was read at 565 nm (SpectraMax® iD3, Molecular Devices, USA).
ALDH inhibition for a test compound is calculated as follows: where ∆OD test compound is the OD 565nm value of test compound minus the OD 565nm value of blank well at 30 min.∆OD no inhibitor is the OD 565 nm value of control minus the OD 565 nm value of blank well at 30 min.

Statistical analysis
All the analyses were carried out in triplicates and reported as mean ± SD.The mean differences in the samples were assessed by Analysis of Variance (ANOVA) with post-hoc analysis using Tukey's test at p < 0.05 using SPSS Statistics 29 software (IBM, USA).